Determination of ß-Galactooligosaccharides (GOS) in Infant Formula: Collaborative Study, Final Action 2021.01.

BACKGROUND: We previously published a method for the determination of ß-Galactooligosaccharides (GOS) in Infant Formula and Adult Nutritionals which is currently First Action AOAC Method 2021.01. In this study, reproducibility data were collected to support the promotion of the method to Final Action. METHOD: A collaborative study was organized, to which 14 laboratories from eight different countries participated. Initially, laboratories were requested to analyze two practice samples and request guidance from the study director in case of issues. Successful laboratories proceeded to analyze seven samples (six infant formula and one adult nutritional) received as blind duplicates. RESULTS: Thirteen laboratories reported acceptable results for practice sample 1. Practice sample 2 could only be delivered to eight of the laboratories due to restrictions at customs. The 13 laboratories successfully analyzing practice sample 1 were requested to continue with the analysis of the MLT samples. Laboratory 14 was unable to solve some technical difficulties, so their data could not be used. Out of the seven samples tested, results for six infant formula met the requirements of the AOAC SMPR 2014.003, with RSDr ranging from 1.4 to 4.7% and RSDR ranging from 8.1 to 11.6%. The adult nutritional sample returned results outside the range of SPMR, having repeatability (RSDr) of 9.9%, higher than the SMPR target of ≤ 6%, and reproducibility (RSDR) of 12.1%, just above the SMPR target of ≤ 12%.

Determination of Seven Human Milk Oligosaccharides (HMOs) in Infant Formula and Adult Nutritionals: First Action 2022.07.

BACKGROUND: Human milk oligosaccharides (HMOs) are important components of breast milk and may be responsible for some of the benefits of breastfeeding, including resistance to infections and the development of a healthy gut microbiota. Selected HMOs are now available for addition to infant formula, and suitable methods to control the dosing rate are needed. OBJECTIVE: To develop and validate a suitable method for the analysis of HMOs in infant formula. METHOD: A method was developed for the determination of seven human milk oligosaccharides (2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose (6'SL), 2',3-difucosyllactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT)) in infant formula and adult nutritionals. The oligosaccharides are labeled at their reducing end with 2-aminobenzamide, separated by liquid chromatography and detected using a fluorescence detector. Maltodextrins are enzymatically hydrolyzed before analysis to prevent potential interference; likewise, an optional ß-galactosidase treatment can be used to remove ß-galactooligosaccharides. Fructooligosaccharides or polydextrose do not generally interfere with the analysis. RESULTS: The method has been validated in a single laboratory on infant formula and adult nutritionals. The seven HMOs were spiked into eight matrixes at three or four spike levels, giving a total of 176 data points. Recoveries were in the range of 90.9-109% in all cases except at the lowest spike level in one matrix (elemental formula), where the LNT recovery was 113%, the LNnT recovery was 111%, and the 6'SL recovery was 121%. Relative repeatabilities (RSD(r)) were in the range of 0.1-4.2%. The performance is generally within the requirements outlined in the Standard Method Performance Requirements (SMPR®) published by AOAC INTERNATIONAL. CONCLUSIONS: The method developed is suitable for the determination of seven HMOs in infant formula and demonstrated good performance during single-laboratory validation. HIGHLIGHTS: A method has been developed that is suitable for the determination of seven HMOs in infant formula.

Determination of ß-Galactooligosaccharides (GOS) in Infant Formula and Adult Nutritionals: Single-Laboratory Validation, First Action 2021.01.

BACKGROUND: ß-Galactooligosaccharides (GOS) are typically used in infant formula and adult nutritionals as a source of nondigestible oligosaccharides, which may bring beneficial effects through modulation of the gut microbiota. However, suitable methods for the determination of GOS in products with a high background of lactose do not exist. OBJECTIVE: The aim of this work was to develop a method suitable for the determination of GOS in infant formula and adult nutritionals and demonstrate suitability through single laboratory validation. METHODS: Reducing oligosaccharides are labeled with 2-aminobenzamide (2AB), separated by hydrophilic interaction LC, and determined assuming that all oligosaccharides give an equimolar response in the detector. The same sample is analyzed a second time after treatment with ß-galactosidase to remove GOS. The difference in the determined oligosaccharides between the two measurements will be the GOS content of the sample. The method was validated in a single laboratory on infant formula and adult nutritionals. RESULTS: Recoveries were in the range 91.5-102%, relative standards of deviation (RSDr) were in the range 0.7-5.99%, and one sample had an RSDr of 8.30%. Except for the one sample with an RSDr of 8.30%, the performance is within the requirements outlined in the Standard Method Performance Requirements, which specifies recoveries in the range 90-110% and RSDr of below 6%. CONCLUSIONS: The method is suitable for the determination of GOS in infant formula and adult nutritionals. HIGHLIGHTS: A method has been developed which is suitable for the determination of GOS in products with a high background concentration of lactose (infant fromula and adult nutritionals). The method does not require access to the GOS ingredient used for the production of the finished product. It is also possible to separately quantify the amount of GOS containing three or more monomeric units in order to support dietary fibre analysis.

Comparison of macronutrient content in human milk measured by mid-infrared human milk analyzer and reference methods.

OBJECTIVE: The study aims at evaluating mid-infrared human milk analyzer (HMA) accuracy and precision, in human milk (HM). STUDY DESIGN: Röse-Gottlieb, high-performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), Kjeldahl and amino acid analysis (AA) were selected as references for total fat, lactose and total protein determination. RESULTS: No significant difference was observed in lactose content between HMA and HPAEC-PAD. Significant differences were observed in fat and protein content between HMA and reference methods. However, the difference in fat content was lower than 12%, and therefore within the variability declared by supplier. For protein determination, the BCA protein assay was selected. No significant differences were observed in total protein content measured by BCA assay, Kjeldahl and AA methods. CONCLUSIONS: HMA was reliable for the quantification of total fat and lactose content, but not for total protein one. The latter was measured by BCA assay, which yielded comparable results to Kjeldahl and AA methods.

Determination of 2'-Fucosyllactose and Lacto-N-neotetraose in Infant Formula.

Human milk oligosaccharides (HMO) are the third most abundant solid component of human milk. It is likely that they are responsible for at least some of the benefits experienced by breast-fed infants. Until recently HMO were absent from infant formula, but 2'-fucosyllactose (2'-FL) and lacto-N-neoteraose (LNnT) have recently become available as ingredients. The development of formula containing these HMO and the quality control of such formula require suitable methods for the accurate determination of the HMO. We developed two different approaches for analysis of 2'-FL and LNnT in formula; high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD). In lab trials using blank formula spiked with the two oligosaccharides, both approaches worked well with recoveries of 94⁻111% (HPAEC-PAD) and 94⁻104% (HILIC-FLD) and RSD (iR) of 2.1⁻7.9% (HPAEC-PAD) and 2.0⁻7.4% (HILIC-FLD). However, when applied to products produced in a pilot plant, the HPAEC-PAD approach sometimes delivered results below those expected from the addition rate of the ingredients. We hypothesize that the oligosaccharides interact with the formula matrix during the production process and, during sample preparation for HPAEC-PAD those interactions have not been broken. The conditions required for labeling the HMO for detection by the FLD apparently disrupt those interactions, and result in improved recoveries. It is likely that both analytical approaches are appropriate if a suitable extraction process is used to recover the HMO.

Determination of ß -Galactooligosaccharides by Liquid Chromatography.

Beta-galactooligosaccharides (GOS) are oligosaccharides normally produced industrially by transgalactosylation of lactose. They are also present naturally in the milk of many animals including humans and cows. GOS are thought to be good for health, being potential prebiotic fibres, and are increasingly added to food products. In order to control the GOS content of products, the AOAC official method 2001.02 was developed. However, the method has some shortcomings and in particular is unsuited to the analysis of products containing high levels of lactose such as infant formula. To overcome this problem, we developed a new method for application to infant formula and tested it on various GOS ingredients as well as infant formulae. When applied to GOS ingredients the results of the new method compare well with those of the official AOAC method, typically giving results in the range 90-110% of those of the official method and having an expanded measurement uncertainty of less than 15%. For three products, the results were outside this range (recoveries of 80-120% and expended measurement uncertainties up to 20%). When applied to the analysis of infant formula, recoveries were in the range of 92-102% and the expanded measurement uncertainties were between 4.2 and 11%.

Development and single-laboratory validation of a method for the determination of total fructans in infant formula.

A number of methods have previously been developed for the determination of fructans in food products, resulting in the frequently used AOAC Official Methods 997.08 and 999.03. Method 997.08 has a lower LOQ, but the accuracy may suffer if significant quantities of sucrose are present. Method 999.03 is less affected by sucrose, but the LOQ is not as low as that of the other method. In this work we have combined these two methods, using the sample preparation steps of AOAC Method 999.03 to remove sucrose and other free sugars, and the detection procedure of AOAC Method 997.08 to achieve a low LOQ. The resulting new method achieved an LOQ of 0.13 g/100 g with powdered products (using 1 g sample weight) and recoveries in the range of 83-103% in the presence of sucrose at 12 g/100 g. Expanded measurement uncertainties were calculated and ranged from +/-17.4% at oligofructose and fructooligosaccharide (FOS) concentrations of around 0.2 g/100 g to -/+21.0% at FOS concentrations of 0.6 g/100 g.